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• Studied the impact of biological stimuli on the phase behaviour of PNIPAM. • Biophysical techniques have used to predict the phase transition behaviour. • The phase behaviour of PNIPAM has been altered upon the addition of proteins. • Explored the interactions between thermoresponsive polymer and proteins. • The present study can pave way for drug delivery and pharmaceutical applications. Continuing efforts to develop stimuli-responsive polymers (SRPs) as novel smart materials/biomaterials are anticipated to upgrade the quality life of humans. The details of the molecular, physico chemical and biophysical interactions between SRPs and proteins are not fully understood. Indeed, protein - polymer interactions play a major role in a wide range of biomedical/biomaterial applications. In this regard, we have demonstrated the influence of proteins (β -lactoglobulin (BLG) and stem bromelain (BM) as biological stimuli) on the phase transition behavior of biomedical thermoresponsive poly(N-isopropylacrylamide) (PNIPAM). In order to predict these, we have used a set of biophysical techniques to unveil the influence of biological stimuli on the phase transition behavior of PNIPAM. Absorption spectroscopy, steady-state fluorescence spectroscopy, Fourier transform infrared (FTIR) spectroscopy, field emission scanning electron microscopy (FESEM) were operated at room temperature to examine the changes in absorbance, fluorescence intensity, molecular interactions and surface morphologies, respectively. Furthermore, temperature dependent fluorescence spectroscopy and dynamic light scattering (DLS) studies were also performed to analyze conformational changes, agglomeration behavior, particle size, coil to globule transition and phase behavior. The significant variations obtained in the phase transition temperature values, conformational changes and agglomeration behavior clearly reflects the different molecular interplay induced in presence of biological stimuli. The results demonstrated that the added proteins act as biological stimuli via preferential interactions between the amide group of the polymer and water molecules. The present study can be useful for the design and development of the next generation smart responsive materials/biomaterials. [ABSTRACT FROM AUTHOR]